On-column capping of poly dT media-tethered mRNA accomplishes high capping efficiency, enhanced mRNA recovery, and improved stability against RNase.

The messenger RNA (mRNA) 5'-cap structure is indispensable for mRNA translation initiation and stability. Despite its importance, large-scale production of capped mRNA through in vitro transcription (IVT) synthesis using vaccinia capping enzyme (VCE) is challenging, due to the requirement of tedious and multiple pre-and-post separation steps causing mRNA loss and degradation. Here in the present study, we found that the VCE together with 2'-O-methyltransferase can efficiently catalyze the capping of poly dT media-tethered mRNA to produce mRNA with cap-1 structure under an optimized condition. We have therefore designed an integrated purification and solid-based capping protocol, which involved capturing the mRNA from the IVT system by using poly dT media through its affinity binding for 3'-end poly-A in mRNA, in situ capping of mRNA 5'-end by supplying the enzymes, and subsequent eluting of the capped mRNA from the poly dT media. Using mRNA encoding the enhanced green fluorescent protein as a model system, we have demonstrated that the new strategy greatly simplified the mRNA manufacturing process and improved its overall recovery without sacrificing the capping efficiency, as compared with the conventional process, which involved at least mRNA preseparation from IVT, solution-based capping, and post-separation and recovering steps. Specifically, the new process accomplished a 1.76-fold (84.21% over 47.79%) increase in mRNA overall recovery, a twofold decrease in operation time (70 vs. 140 min), and similar high capping efficiency (both close to 100%). Furthermore, the solid-based capping process greatly improved mRNA stability, such that the integrity of the mRNA could be well kept during the capping process even in the presence of exogenously added RNase; in contrast, mRNA in the solution-based capping process degraded almost completely. Meanwhile, we showed that such a strategy can be operated both in a batch mode and in an on-column continuous mode. The results presented in this work demonstrated that the new on-column capping process developed here can accomplish high capping efficiency, enhanced mRNA recovery, and improved stability against RNase; therefore, can act as a simple, efficient, and cost-effective platform technology suitable for large-scale production of capped mRNA.

Moderate anthropogenic disturbance stimulates versatile microbial taxa contributing to denitrification and aromatic compound degradation.

Wastewater treatment plants (WWTPs) effluent often contains a significant amount of residual organic pollutants and nutrients, causing disturbance to the coastal effluent receiving areas (ERA). Microbial communities in coastal ERA sediments may benefit from the coexistence of organic pollutants and nutrients, promoting the emergence of versatile taxa that are capable of eliminating these substances simultaneously. However, the identification and exploration of versatile taxa in natural environments under anthropogenic disturbances remain largely uncharted territory. In this study, we specifically focused on the versatile taxa coupled by the degradation of aromatic compounds (ACs) and denitrification, using Hangzhou Bay in China as our study area. We explored how WWTPs effluent disturbance would affect the versatile taxa, and particularly examined the role of disturbance intensity in shaping their composition. Intriguingly, we found that versatile taxa were mainly derived from denitrifiers like Pseudomonas, suggesting the fulfilled potential of denitrifiers regarding ACs degradation. We also discovered that moderate disturbance stimulated the diversity of versatile taxa, resulting in strengthened functional redundancy. Through correlation network analysis, we further demonstrated that moderate disturbance enhanced the community-level cooperation. Thus, moderate disturbance serves as a catalyst for versatile taxa to maintain community function, making them more resilient to effluent disturbances. Additionally, we identified COD and NO3--N concentrations as significant environmental factors influencing the versatile taxa. Overall, our findings reveal the role of effluent disturbances in the promotion of versatile taxa, and highlight moderate disturbance can foster more robust versatile taxa that are better equipped to handle effluent disturbances.

A New Technique for Chondrolaryngoplasty in Patients Assigned Female at Birth.

BACKGROUND: Chondrolaryngoplasty is a classical facial feminization surgery for transgender women. In recent years, however, an increasing number of patients assigned female at birth are seeking chondrolaryngoplasty for esthetic purposes. Traditional chondrolaryngoplasty can no longer cope with problems of the growing group whose leading cause of laryngeal prominence differs from the transgender population. METHODS: A modified technique is designed as a supplement to the classical procedure. After the cartilage reduction process, paired platysma flaps are raised and advanced successively, resulting in an overlapped area over the thyroid notch, to further camouflage the thyroid prominence. To evaluate the efficiency of the new technique, a retrospective survey of 34 patients (5 men and 29 women) who underwent the surgery from 2016 to 2021 was performed, via a 5-point Likert scale including 7 questions. Physician assessment was also accomplished to provide an extra estimation. Complications were followed up and analyzed to evaluate the safety of modified surgery as well. RESULTS: Although only half of the patients graded prominence changes more than "moderately changed," as many as 75.0% of them still expressed "completely satisfied" or "satisfied very much" with the outcome. Similarly, physician assessment indicated a satisfactory result in appearance improvement. No severe and irreversible complications occurred after surgery, but lasting scar-related issues were reported by 4 patients and should be paid more attention to. CONCLUSIONS: Generally speaking, the new technique is both safe, efficient, and satisfying for most patients, especially ones assigned females at birth with esthetic demand.

PTB Regulates the Metabolic Pathways and Cell Function of Keloid Fibroblasts through Alternative Splicing of PKM.

Keloids, benign fibroproliferative cutaneous lesions, are characterized by abnormal growth and reprogramming of the metabolism of keloid fibroblasts (KFb). However, the underlying mechanisms of this kind of metabolic abnormality have not been identified. Our study aimed to investigate the molecules involved in aerobic glycolysis and its exact regulatory mechanisms in KFb. We discovered that polypyrimidine tract binding (PTB) was significantly upregulated in keloid tissues. siRNA silencing of PTB decreased the mRNA levels and protein expression levels of key glycolytic enzymes and corrected the dysregulation of glucose uptake and lactate production. In addition, mechanistic studies demonstrated that PTB promoted a change from pyruvate kinase muscle 1 (PKM1) to PKM2, and silencing PKM2 substantially reduced the PTB-induced increase in the flow of glycolysis. Moreover, PTB and PKM2 could also regulate the key enzymes in the tricarboxylic acid (TCA) cycle. Assays of cell function demonstrated that PTB promoted the proliferation and migration of KFb in vitro, and this phenomenon could be interrupted by PKM2 silencing. In conclusion, our findings indicate that PTB regulates aerobic glycolysis and the cell functions of KFb via alternative splicing of PKM.

Possible Action of Transition Divalent Metal Ions at the Inter-Pentameric Interface of Inactivated Foot-and-Mouth Disease Virus Provide A Simple but Effective Approach to Enhance Stability.

The structural instability of inactivated foot-and-mouth disease virus (FMDV) hinders the development of vaccine industry. Here we found that some transition metal ions like Cu2+ and Ni2+ could specifically bind to FMDV capsids at capacities about 7089 and 3448 metal ions per capsid, respectively. These values are about 33- and 16-folds of the binding capacity of non-transition metal ion Ca2+ (about 214 per capsid). Further thermodynamic studies indicated that all these three metal ions bound to the capsids in spontaneous enthalpy driving manners (ΔG<0, ΔH<0, ΔS<0), and the Cu2+ binding had the highest affinity. The binding of Cu2+ and Ni2+ could enhance both the thermostability and acid-resistant stability of capsids, while the binding of Ca2+ was helpful only to the thermostability of the capsids. Animal experiments showed that the immunization of FMDV bound with Cu2+ induced the highest specific antibody titers in mice. Coincidently, the FMDV bound with Cu2+ exhibited significantly enhanced affinities to integrin ß6 and heparin sulfate, both of which are important cell surface receptors for FMDV attaching. Finally, the specific interaction between capsids and Cu2+ or Ni2+ was applied to direct purification of FMDV from crude cell culture feedstock by the immobilized metal affinity chromatography. Based on our new findings and structural analysis of the FMDV capsid, a "transition metal ion bridges" mechanism that describes linkage between adjacent histidine and other amino acids at the inter-pentameric interface of the capsids by transition metal ions coordination action was proposed to explain their stabilizing effect imposed on the capsid.IMPORTANCE How to stabilize the inactivated FMDV without affecting virus infectivity and immunogenicity is a big challenge in vaccine industry. The electrostatic repulsion induced by protonation of a large amount of histidine residues at the inter-pentameric interface of viral capsids is one of the major mechanisms causing the dissociation of capsids. In the present work, this structural disadvantage inspired us to stabilize the capsids through coordinating transition metal ions with the adjacent histidine residues in FMDV capsid, instead of removing or substituting them. This approach was proved effective to enhance not only the stability of FMDV, but also enhance the specific antibody responses; thus, providing a new guideline for designing an easy-to-use strategy suitable for large-scale production of FMDV vaccine antigen.

Low-level laser treatment promotes skin wound healing by activating hair follicle stem cells in female mice.

The aim of the study was to explore the effect and mechanism of a low-level laser on hair follicle stem cells in full-thickness skin wound healing in mice. Full-thickness skin defects were generated by a 5-mm punch biopsy tool on the backs of depilated C57/BL6N mice, which were randomly divided thereafter into a low-dose laser treatment group (LLLT-Low), a high-dose laser treatment group (LLLT-High), and a control group (control). From the day of modeling to the day before the skin samples were taken, the wound area and wound edge of the mice in the LLLT-Low and LLLT-High groups were irradiated with a laser comb every 24 h, and the energy density was 1 J/cm2 and 10 J/cm2, respectively. The control group was irradiated with an ordinary fluorescent lamp. At 0, 3, 5, 10, and 14 days after modeling, pictures of each wound were taken, and the percent wound closure was analyzed. At 3, 5, 10, and 14 days after modeling, the samples were observed by hematoxylin and eosin (HE) and immunofluorescence (IF) staining. Whole transcriptome sequencing (RNA-Seq) was performed on the samples on day 10. Gene Ontology (GO) analysis was performed, and the results were validated by Western blot analysis and enzyme-linked immunosorbent assay (ELISA). The analysis of the percent of wound closure showed that healing was accelerated (significantly from 5 to 10 days) in the LLLT-Low group, but there was no clear change in the LLLT-High group. HE staining showed that the LLLT-Low group had an increasing number of hair follicles and a tendency to migrate to the center of the wound. There was no significant increase in the number of hair follicles and no obvious migration in the LLLT-High group. Immunofluorescence staining showed that the total number of CK15 + hair follicle stem cells in the LLLT-Low group was higher than that in the control group and LLLT-High group at all time points. The number and farthest migration distance of CK15 + hair follicle stem cells increased significantly with time, and after 5 days, they were significantly higher than those in the control group and LLLT-High group. RNA-Seq and Western blot analysis showed that the expression of related genes in hair follicle stem cells, including CK15, in the LLLT-Low group was upregulated. GO analysis and ELISA showed that the expression of many cytokines, represented by IL34, in the LLLT-Low group was upregulated. Low-level laser treatment can promote the proliferation, differentiation, and migration of CK15 + hair follicle stem cells by upregulating the cytokine IL34, thereby promoting skin wound healing in mice.

Three-Dimensional Z-Epicanthoplasty Retains the Depth of the Medial Canthus With Scars That Are Less Visible.

BACKGROUND: Traditional coronal Z-epicanthoplasty usually generates visible scars, and V-Y advancement or skin redraping can damage the normal shape of the lower eyelid. In addition, these methods usually lead to loss of the medial canthus depth, which is important for the natural appearance of East Asians. This report aims to describe a 3-dimensional Z-epicanthoplasty that can maintain the depth of the medial canthus with fewer visible scars. METHODS: Patients who underwent 3-dimensional Z-epicanthoplasty from January 2016 to December 2019 were included. In our method, excess skin in the horizontal direction was first rotated to the vertical position and then turned inward from a coronal to a sagittal orientation to supplement the skin deficiency of the medial canthus in the sagittal orientation. The skin flap turned in the sagittal orientation and maintained the depth of the medial canthus and the natural appearance of the eye. RESULTS: In total, 138 patients were included, and the follow-up interval ranged from 6 to 36 months. After the surgery, the epicanthi were removed, and the shapes of the patients' eyes were significantly improved and appeared natural. The surgical scars were hidden in the sagittal orientation rather than in the coronal orientation, which cannot be seen easily. The depth of the medial canthus was well preserved without any lower eyelid destruction. Patients were satisfied with the natural appearance of the medial canthus. CONCLUSIONS: This method could not only correct the epicanthal fold effectively with scars that are less visible but also maintain the depth of the medial canthus and make the appearance of the eye look more natural.

Inhibiting Warburg Effect Can Suppress the Biological Activity and Secretion Function of Keloid Fibroblasts.

BACKGROUND: Keloids have always been a difficult problem in the clinic. In our previous study, we demonstrated a Warburg effect in keloid fibroblasts (KFs), like tumors. In this study, we aimed to investigate the effects of the suppression of the Warburg effect on the biological activity and function of KFs. METHODS: KFs were isolated and cultured with different concentrations of oxamate, a classical competitive lactate dehydrogenase A (LDHA) inhibitor. First, the suppression effect of oxamate on the Warburg effect in KFs was verified. After treatment with oxamate, a scratch wound assay, real-time PCR, flow cytometry, CCK8 kit, and western blotting were used to detect the migration ability, collagen production, apoptosis, cell proliferation, cell cycle distribution, and related molecular mechanisms in KFs. RESULTS: As expected, oxamate inhibited the Warburg effect in KFs in a dose-dependent manner. After the inhibition of the Warburg effect in KFs, the cell migration rate decreased significantly, the mRNA transcription levels of type I collagen and α-SMA were significantly lower, the cell apoptosis rate increased significantly, the cell proliferation activity decreased significantly, and G0/G1 phase cells in KFs increased significantly. The expression of cyclin D1 and its upstream regulatory factors, Akt protein and GSK3 ß (phospho S9), decreased significantly. CONCLUSION: Inhibiting the Warburg effect in KFs significantly suppressed cell proliferation, enhanced cell apoptosis, inhibited cell migration ability, reduced collagen secretion, and induced G0/G1 arrest through the Akt-GSK3ß-Cyclin D1 pathway. Therefore, inhibiting the Warburg effect in KFs may provide a new option for the prevention and treatment of keloids. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors   www.springer.com/00266 .

In vitro preparation of uniform and nucleic acid free hepatitis B core particles through an optimized disassembly-purification-reassembly process.

Structure heterogeneity and host nucleic acids contamination are two major problems for virus-like particles (VLPs) produced by various host cells. In this study, an in vitro optimized disassembly-purification-reassembly process was developed to obtain uniform and nucleic acid free hepatitis B core (HBc) based VLPs from E. coli fermentation. The process started with ammonium sulfate precipitation of all heterogeneous HBc structures after cell disintegration. Then, dissolution and disassembly of pellets into basic subunits were carried out under the optimized disassembly condition. All contaminants, including host nucleic acids and proteins, were efficiently removed with affinity chromatography. The purified subunits reassembled into VLPs by final removal of the chaotropic agent. Two uniform and nucleic acid free HBc-based VLPs, truncated HBc149 and chimeric HBc183-MAGE3 I, were successfully prepared. It was found that disassembly degree of HBc-based VLPs had a great influence on the protein yield, nucleic acid removal and reassembly efficiency. 4 M urea was optimal because lower concentration would not disassemble the particles completely while higher concentration would further denature the subunits into disordered aggregate and could not be purified and reassembled efficiently. For removal of strong binding nucleic acids such as in the case of HBc183-MAGE3 I, benzonase nuclease was added to the disassembly buffer before affinity purification. Through the optimized downstream process, uniform and nucleic acid free HBc149 VLPs and HBc183-MAGE3 I VLPs were obtained with purities above 90% and yields of 55.2 and 43.0 mg/L, respectively. This study would be a reference for efficient preparation of other VLPs.

The Effects of Inactive Platelet-rich Plasma at Different Injection Time on Prefabricated Flap Viability in Rabbits.

PURPOSE: To evaluate the effect of inactive form of platelet-rich plasma (PRP) at different injection time on the prefabricated flap viability in rabbits. METHODS: A thoracodorsal artery vascular bundle prefabricated flap was established on the back of 30 healthy male New Zealand white rabbits by 2 phases of operations. A total of 60 sides were randomly divided into the experimental side and the control side. The experimental side was injected with autologous inactive PRP around the thoracodorsal vascular bundle, and the control side was injected with the same amount of normal saline. According to preoperative (group A), intraoperative (group B), and postoperative (group C) injection of PRP, 30 rabbits were divided into 3 groups of 10 each. Histological and immunohistochemical observation, vascular density measurement, and lead oxide angiography were used to compare the differences in the survival of prefabricated flaps between the 3 groups of rabbits injected with inactive PRP at different injection time. RESULTS: The flap survival ratio, microvessels density, vascular endothelial growth factor and vascular endothelial growth factor receptor 2 expression of PRP injection side were significantly improved compared with the control side. In the comparison of 3 injection time groups, we found that group A and group B could achieve better results, and especially group B had the most significant effect on the revascularization and flap viability. CONCLUSION: Autologous inactive PRP can promote the survival of the prefabricated flap, and the intraoperative injection can significantly improve the survival rate of the prefabricated flap. It provides a theoretical guidance for obtaining a larger area of prefabricated axial flap in clinical practice.

"Zipper" Concept for Designing Expanded Forehead Flap in Facial-Cervical Reconstruction.

BACKGROUND: Skin defect of the lower part of the face and neck is a severe condition for many patients with burn and traumatic injuries or skin lesions. The expanded forehead flap with anterofrontal temporal vessel pedicles has been widely used in lower cheeks, chin, and neck reconstruction. (In this study, we introduce a new concept- "Zipper concept" of the flap design.) The aim of this study is to evaluate the feasibility and outcome of using expanded forehead flap with a vertical incision with or without a small transverse incision in the central part. METHODS: From March 2010 to September 2016, 27 patients who suffered from lower cheeks and chin skin defects were reconstructed by expanded forehead flap with anterofrontal temporal vessel pedicles. After the lesion was excised, the upper or the lower part of the flap was cut in half with a vertical incision with or without a small transverse incision in the middle part as needed for cheeks, upper lip or neck reconstruction, and the other part of the flap was remained to be one piece for chin reconstruction. RESULTS: All flaps survived completely without any congestion or necrosis. All patients achieved satisfactory results both functionally and aesthetically. CONCLUSIONS: A vertical incision with or without a small transverse incision in the central part of the expanded forehead flap with anterofrontal temporal vessel pedicles is a safe modification of the flap design. It is practical and could bring the expanded forehead flap to more indications.

Nanofat Injection for the Treatment of Depressed Facial Scars.

BACKGROUND: Patients with depressed facial scars complain of their negative effects. However, the efficacy of optional treatment techniques is never completely adequate. This study aimed to assess the efficacy of nanofat injection in the improvement of depressed facial scars. METHODS: This retrospective study included patients who underwent depressed facial scar filling with nanofat between November 2017 and January 2020. The FACE-Q scale was sent to patients for feedback regarding satisfaction. Evaluations of the results were also performed by three plastic surgeons. RESULTS: Among the 52 included patients, 44 patients (29 women and 15 men) completed the questionnaire. Obvious and stable effects were usually acquired 3 months after surgery. Temporary erythema appeared at the injection site to varying degrees, lasting 2 to 3 weeks in 93% of the patients. No other serious postoperative complications were observed in the injection area. The FACE-Q outcomes showed that patients who completed injection therapy more than 1 year prior were significantly more satisfied with the decision to undergo this therapy than those who completed the treatment less than 1 year prior. Furthermore, according to the physicians' evaluations, 91% of patients experienced improvement in scar appearance after treatment. CONCLUSIONS: The low rate of injection-site complications and the safety of this procedure both support the current implementation of nanofat in the treatment of depressed facial scars. The results of the physicians' evaluations and patient satisfaction surveys confirmed the stable effect of nanofat injection in the treatment of depressed facial scars. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

The Application of Temporal-Fasciae-Complex Sheet in Treating Severe Blepharoptosis.

AIM: To introduce the application of the temporal-fasciae-complex sheet in treating severe blepharoptosis by frontalis suspension and evaluate its postoperative effect. METHODS: Between 2008 and 2016, 25 patients (33 eyelids) underwent this procedure. A 3-cm incision in the temporal region was made to harvest a sheet of deep temporal fascia with the loose aponeurosis attached on both sides. The sheet was then grafted through a preseptal tunnel to perform the suspension. The margin reflex distance 1 after suspension (MRD1S), the margin reflex distance 1 as lifting eyebrow forcefully (MRD1F), the eyelid excursion and the closable eyelid function were used to evaluate the postoperative effect. RESULTS: A total of 22 patients (30 eyelids) completed the study with a mean follow-up period of 23 ± 8.78 months. There was a statistically significant difference between the MRD1 and MRD1S (p < 0.05), the preoperative and postoperative MRD1F (p < 0.05), the preoperative and postoperative eyelid excursion (p < 0.05). All the upper palpebral margins were located above the pupils and no longer affected visual acuity in primary gaze. No severe complication and recurrence were documented within a maximum follow-up period of 36 months. CONCLUSIONS: Frontalis suspension with the temporal-fasciae-complex sheet is an efficient method to correct severe blepharoptosis with less complications and recurrences. The application of the sheet can not only overcome the influence of adhesion but also lift the eyelid both functionally and cosmetically. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

An Apoferritin-Hemagglutinin Conjugate Vaccine with Encapsulated Nucleoprotein Antigen Peptide from Influenza Virus Confers Enhanced Cross Protection.

Naturally occurring self-assembling ferritin nanoparticles have become widely appreciated for vaccine design. In this study, an apoferritin (AFt) nanocage was used as a carrier to construct a biomimetic influenza vaccine by encapsulating a conserved internal nucleoprotein (NP) antigen peptide inside the nanocage, followed by chemically conjugating the surface antigen hemagglutinin (HA) protein on the outer surface of the AFt. Benefiting from the excellent thermal stability and thermallyassociated structural flexibility of the AFt nanocages, a novel temperature shift based encapsulation process was proposed and proved efficient for encapsulation of the NP peptides. On average, about 18 NPs were encapsulated and 1.6 HA antigens were conjugated in each of the HA-AFt+NP dual-antigen influenza vaccines. Upon immunization in mice, the HA-AFt+NP vaccine elicited both HA and NP-specific antibodies, and conferred complete protection against a lethal infection of both homologous PR8 H1N1 and heterologous A/FM/1/47 (FM1, H1N1) strains, while the HA-AFt conjugate vaccine without encapsulated NP antigen only conferred 60% protection against the FM1 H1N1 viral challenge. The potential cross-protective effect of the HA-AFt+NP vaccine was further demonstrated by significant specific hemagglutination inhibition (HAI) titers in serum of the immunized mice against heterologous A/Hong Kong/4801/2014 (H3N2) viral strain, which was about 3-fold of that induced by HA antigen and 2-fold of the HA-AFt conjugate vaccine. This biomimetic HA-AFt+NP conjugate vaccine, therefore, may represent a new strategy for developing a potential universal influenza vaccine without the need of any adjuvant, and further broaden the application of AFt nanocages in the areas of vaccine development and delivery system.

A Novel Particulate Delivery System Based on Antigen-Zn2+ Coordination Interactions Enhances Stability and Cellular Immune Response of Inactivated Foot and Mouth Disease Virus.

The interactions between antigen and adjuvant were among the most significant factors influencing the immunogenicity of vaccines, especially for unstable antigens like inactivated foot and mouth disease virus (iFMDV). Here we propose a novel antigen delivery pattern based on the coordination interaction between transition metal ions Zn2+ chelated to chitosan nanoparticles and iFMDV, which is known to be rich in histidine. The zinc chelated chitosan particles (CP-PEI-Zn) were prepared by cross-linking chitosan particles (CP) with sodium tripolyphosphate (TPP), modifying with metal chelator polyethylenimine (PEI), and subsequent chelating of Zn2+. The coordination interaction was confirmed by analyzing the adsorption and desorption behavior of iFMDV on CP-PEI-Zn by high-performance size exclusion chromatography (HPSEC), while the CP-PEI without chelating Zn2+ loads iFMDV mainly through electrostatic interactions. The iFMDV loaded on CP-PEI-Zn showed better thermal stability than that on CP-PEI, as revealed by a slightly higher transition temperature (Tm) related to iFMDV dissociation. After subcutaneous immunization in female Balb/C mice, antigens loaded on CP-PEI and CP-PEI-Zn all induced higher specific antibody titers, better activation of B lymphocytes, and more effector-memory T cells proliferation than the free antigen and iFMDV adjuvanted with ISA 206 emulsion did. Moreover, CP-PEI-Zn showed superior efficacy to CP-PEI in promoting the proliferation of effector-memory T cells and secretion of cytokines, indicating a more potent cellular immune response. In summary, the CP-PEI-Zn stabilized the iFMDV after loading and promoted both humoral and cellular immune responses, thus reflecting its potential to be a promising adjuvant for the iFMDV vaccine and other unstable viral antigens.

A Tripartite Microbial-Environment Network Indicates How Crucial Microbes Influence the Microbial Community Ecology.

Current technologies could identify the abundance and functions of specific microbes, and evaluate their individual effects on microbial ecology. However, these microbes interact with each other, as well as environmental factors, in the form of complex network. Determination of their combined ecological influences remains a challenge. In this study, we developed a tripartite microbial-environment network (TMEN) analysis method that integrates microbial abundance, metabolic function, and environmental data as a tripartite network to investigate the combined ecological effects of microbes. Applying TMEN to analyzing the microbial-environment community structure in the sediments of Hangzhou Bay, one of the most seriously polluted coastal areas in China, we found that microbes were well-organized into 4 bacterial communities and 9 archaeal communities. The total organic carbon, sulfate, chemical oxygen demand, salinity, and nitrogen-related indexes were detected as crucial environmental factors in the microbial-environmental network. With close interactions with these environmental factors, Nitrospirales and Methanimicrococcu were identified as hub microbes with connection advantage. Our TMEN method could close the gap between lack of efficient statistical and computational approaches and the booming of large-scale microbial genomic and environmental data. Based on TMEN, we discovered a potential microbial ecological mechanism that crucial species with significant influence on the microbial community ecology would possess one or two of the community advantages for enhancing their ecological status and essentiality, including abundance advantage and connection advantage.

Exploiting the pliability and lateral mobility of Pickering emulsion for enhanced vaccination.

A major challenge in vaccine formulations is the stimulation of both the humoral and cellular immune response for well-defined antigens with high efficacy and safety. Adjuvant research has focused on developing particulate carriers to model the sizes, shapes and compositions of microbes or diseased cells, but not antigen fluidity and pliability. Here, we develop Pickering emulsions-that is, particle-stabilized emulsions that retain the force-dependent deformability and lateral mobility of presented antigens while displaying high biosafety and antigen-loading capabilities. Compared with solid particles and conventional surfactant-stabilized emulsions, the optimized Pickering emulsions enhance the recruitment, antigen uptake and activation of antigen-presenting cells, potently stimulating both humoral and cellular adaptive responses, and thus increasing the survival of mice upon lethal challenge. The pliability and lateral mobility of antigen-loaded Pickering emulsions may provide a facile, effective, safe and broadly applicable strategy to enhance adaptive immunity against infections and diseases.

Efficacy of endovascular intervention in patients with unruptured posterior communicating artery aneurysm-related oculomotor nerve palsy.

OBJECTIVES: Oculomotor nerve palsy (ONP) is commonly encountered in daily neurosurgical activities. The ONP secondary to un-ruptured PComA aneurysm might be a unique entity that was different in diagnosis, treatment and prognosis from its ruptured counterparts. Perhaps as a result of the limitation in sample size, studies that solely focused on factors affecting recovery of ONP in patients with unruptured corresponding PComA aneurysms were scarce. METHODS: In this study, we would like to report a relatively larger case series of patients with un-ruptured PComA aneurysm-related ONP. A retrospective review of medical records of 39 patients with un-ruptured PComA aneurysm-related ONP was performed with endovascular coiling. RESULTS: All 39 consecutive patients underwent endovascular coiling. Eighteen (46%) patients had a complete resolution of ONP, 14 (36%) patients had a partial resolution. Time interval from onset of ONP to endovascular intervention (P=0.004), degree of ONP (P=0.015) and age (P=0.016) were predictors of ONP recovery with statistical significance. Sex, aneurysm size and risk factor exposure (smoking, alcohol abuse and hypertension) were not associated with ONP outcomes. CONCLUSION: ONP secondary to un-ruptured aneurysm should be treated as a unique entity from its ruptured counterparts. A prospective study that contains surgical clipping and endovascular coiling, and comparison between two treatment modalities would be more convincing and is anticipated.

Occurrence and distribution of antibiotic resistance genes in the sediments of the East China Sea bays.

The coastal area of the East China Sea has experienced rapid urbanization and industrialization in China since 1980s, resulting in severe pollution of its environments. Antibiotic resistance genes (ARGs) are regarded as a kind of emerging pollutant with potential high risk. The sediment samples were collected from Hangzhou Bay (HB), Xiangshan Bay (XB), and Taizhou Bay (TB) to investigate the spatial occurrence and distribution of 27 ARGs and class I integron-integrase gene (intI1) in the coastal area of the East China Sea. The PCR results showed the frequent presence of 11 ARGs and intI1 in the sediments of the three bays. The qPCR results further showed that sulfonamide resistance was the most prevalent ARG type and antibiotic target replacement and protection were the most important resistance mechanisms in the sediments. Regarding the subtype of ARGs, sulI, tetW, and dfrA13 were the most abundant ARGs, in which sulI was higher in TB (based on both the absolute and relative abundances) and dfrA13 was higher in HB (based on the relative abundances). The network analysis revealed that intI1 had significant correlations with tetC, sulI, sulII, and blaPSE-1. Oil was the key connected factor, which had positive connections with sulI, sulII, and blaPSE-1. In addition, the joint effect of heavy metals and nutrients & organic pollutants might be crucial for the fate of ARGs in the coastal sediments.

Prompt and Robust Humoral Immunity Elicited by a Conjugated Chimeric Malaria Antigen with a Truncated Flagellin.

As one of the pathogen-associated molecular patterns (PAMPs), flagellin is recently utilized as a potent adjuvant for many subunit vaccines. In this study, a truncated flagellin (tFL) with deletion of the hypervariable regions was adopted as a carrier-adjuvant by chemical conjugation with a chimeric malaria antigen M.RCAg-1 (M312) via a heterobifunctional polyethylene glycol (PEG) linker. After booster immunization in mice without any extra adjuvants, the M312-PEG-tFL conjugates elicited M312-specific antibody titers 100-1000 times higher than M312 and 10-100 times higher than the physical mixture of M312 and tFL. The elicited specific antibodies could recognize the native parasites, and the immunofluorescence assay (IFA) titer was 2100 for M312-P5k-tFL, which was about 7 times higher than M312. Furthermore, the IFA titers of the conjugates were comparable to the positive control of complete Freund's adjuvant (CFA). Compared to M312, the M312-PEG-tFL conjugates enhanced the proliferation index, lymphocyte activation, and memory T-cell generation. IgG subclasses of sera and cytokines analysis of splenocytes showed that conjugation with tFL could slightly trigger the Th1 polarization, while the antigen alone predominantly induced a Th2-biased immune response. Furthermore, a more-efficient innate immune response was provoked by the M312-PEG-tFL conjugates, as determined by the detection of antigen-specific TNF-α secretion by splenocytes. Our results indicated that tFL mainly retained the function as an agonist of TLR5. Conjugation of antigen to tFL could induce strong humoral and moderate cellular immune responses. Thus, conjugation of antigen to tFL as a potent carrier-adjuvant is an effective strategy for developing a promising protein-based vaccine.